Phosphonates, biphosphonates and pharmaceutical compositions containing them

ABSTRACT

A method for treating patients having calcium related disorders includes using di- or tripeptide derivatives of bisphosphonates to enhance oral bioavailability of such compounds.

BACKGROUND OF THE INVENTION

Calcium-related disorders in general and osteoporosis in particular area major public health problem in developed countries. Several importantpathological conditions are calcium-related and involve irregularitiesin calcium metabolism: Paget's disease, osteoporosis, hypercalcemia ofmalignancy, and osteolysis front bone metastases, etc. Bisphosphonatesare a relatively new family of drugs used clinically in variouscalcium-related disorders including tumor osteolysis, and are undergoingclinical trials for osteoporosis. They are poorly absorbed followingoral administration probably due to their high polarity and charge. IVand IM administration is a serious obstacle to their wide-spread use.

In the patent of Bosies et al. (U.S. Pat. No. 4,666,895 May 19, 1987)peptidyl diphosphonic acid derivatives are described. Bosies et al.Patent hypothesize that some peptidyl compounds will have betteractivity on bone: “In particular, they can be used in cases where theformation and breakdown of bone is disturbed, for example in cases ofosteoporosis”. Nothing is claimed, regarding the absorption of suchcompounds from the gastrointestinal tract following oral administration.The dosage recommended is much higher than that needed when effectiveoral absorption takes place. The dosage range recommended (see column 6)is from . . . “1 mg to 1000 mg, and preferably from 10 to 200 mg.” Suchdoses are typical for non-absorbable bisphosphonates such as etidronateand pamidronate. The daily oral recommended dose of a recently approvedbisphosphonate in the US, alendronate (Fosamax) is 10 mg and 40 mg, inthe treatment of osteoporosis and Paget's disease, respectively(American Hospital Formulary Service AHFS Drug Information, section92:00, 1995). As is known widely, this drug is 100 to 1000 times moreeffective than etidronate, the drug chosen by Bosies et al, forcomparison. Typical absorption of such bisphosphonates in humans is inthe range of 1%. Effective hydrolysis of the prodrug to the parent drug,for e.g., pamidronate or alendronate, is achieved following oraladministration in the cytosol (intestinal cells). It is clear that if apeptidyl derivative of such a drug is administered to humans the doseshould be reduced by 50 to 100 times, in order not to be lethal, due tothe enhanced absorption of about 50 to 100 times of the prodrug. Bosiesrelates only to subcutaneous injections, in comparison to a firstgeneration bisphosphonate, etidronate. Furthermore, selection of thepeptidyl derivatives is not based on recognition by the transportersystem nor on the possibility for hydrolysis to the parent drug. Intheir patent they seed a new compound, the administered drug being theactive drug at the site of action (bone). The present invention relatesspecifically to the activity of the parent drug yielded form the prodrugfollowing oral absorption.

The results are described ambiguously (+ and −system), and the activityof the compounds is compared to first generation bisphosphonate,etidronate (a non-nitrogen containing compound) rather than a moreappropriate comparison to nitrogen-containing drugs such as pamidronate,known to be more effective (pamidronate and alendronate, nitrogencontaining bisphosphonates, in clinical use are 10 to 1000 times moreeffective than etidronate. To the best of our knowledge no furtherdevelopment (since 1987) nor a scientific report is available for suchcompounds claimed by Bosies et al.

Bisphosphonates

Bisphosphonates have been approved for clinical use in Paget's disease,tumor osteolysis, and hypercalcemia of malignancy and approved in somecountries for osteoporosis therapy. Most bisphosphonates are disodiumsalts of tie tetraacids (M.W. approximately 250), and are poorlyabsorbed from the GI tract (approximately 1% of the oral dose isabsorbed). Chronic IM or SC administration of bisphosphonates causesirritation and necrosis, and the oral route has been associated with GIdisturbances, resulting in poor patient compliance and side effects. Forexample, the treatment protocol of pamidronate in tumor osteolyis is1-day slow and diluted IV infusion to avoid thrombophlebitis, buttreatment is repeated if normocalcemia is not attained. Another exampleis the chronic therapy (years) required in osteoporosis.

Absorption Barriers

Clinically, the oral route is the most common and accepted one fordelivering drugs of a low molecular weight, of up to 400-600. However,the low permeability of the intestinal epithelia towards highly polarand charged molecules impedes the effective absorption of many lowmolecular weight drugs. Many such drugs must be delivered parenterallyby frequent injections. This is highly risky without close medicalsupervision. The problem is particularly acute in cases of drugs usedfor treatment of various chronic diseases such as cancer and age-relateddiseases, such as osteoporosis, which require prolonged drug treatment.

New Drugs for Calcium-related Disorders

Drugs require a degree of lipophilicity to pass through the GI barrier.In order to increase oral absorption of drugs with low membranepermeability, nonpolar prodrugs are often utilized. Due to the widevariety of esterases present in the target tissue for oralprodrug-regeneration, esters are the most common prodrugs when GIabsorption is considered. Acyloxymethyl esters of bisphosphonic acidswere proposed however this did not lead to a useful drug (EuropeanPatent EP 0 416 689 A2, date of filing Aug. 29, 1990). Similarly, Felset al. proposed pharmaceutical compositions comprising bisphosphonatesand sodium lauryl sulfate for increased oral absorption (U.S. Pat. No.4,980,171, Dec. 25, 1990).

One way to increase membrane permeability of drugs is by utilization ofthe peptide carrier system (G. L. Amidon et al., Absorption of difficultdrug molecules: Carrier-mediated transport of peptides and peptideanalogues, Novel drug delivery and its therapeutic application, L. F.Prescott and W. S. Nimmo, Eds., John Wiley & Sons (1989) pp. 45-56).

SUMMARY OF INVENTION

The hypothesis of the present patent is completely different from theworking hypothesis of Bosies et al. By carefully selecting specific diand/or tripeptide derivatives of bisphosphonates, enhanced oralbioavailability can be achieved due to the recognition by the activecarrier transporter of the intestinal mucosae and the hydrolysis to theparent drug following oral administration.

Thus, the prodrug in our invention is a delivery system rather than anew compound for bone diseases. Therefore, the present selection of newcompounds is based on enhanced absorption for the GI tract andhydrolysis to the parent compound resulting in improved oral, clinicaltreatment by a low dose. The selection of the di/tri-peptidyl moiety isbased on transporter recognition and hydrolysis and not, as in Bosies etal patent, on resorbing activity on the bone.

Thus, the present invention relates to novel compounds of the generalformula

wherein Z is

wherein Z¹ and Z² are independently a side chain of an amino acid orhydrogen,

wherein Z⁴ is hydrogen or Z⁴ and Z¹ together with —N—CH—CO represent aproline residue.

where Q is

where Z³ is a bond or a spacer group selected from NH, CO, NHCO,NHCO(CH₂)_(q)—CO, (CH₂)_(r)CO where r is zero or an integer,

and where q is an integer or zero,

m is 2, 3 or 4,

n is greater than or equal to 0

X is —H or —OH,

Y is —H or —NR²R³,

where A designates a 5- or 6-membered heterocyclic ring which contains1, 2 or 3 nitrogen atoms, zero, 1 or 2 oxygen atoms and which maycontain a sulfur atom, which contains up to and including 3 doublebonds, where R² and R³ are independently hydrogen, lower alkyl, loweralkenyl, lower alkoxy, (di)alkylaminoalkyl, alkoxyalkyl and where thering A may be substituted by one or more conventional substituents, andto pharmaceutical compositions of improved absorption from thegastro-intestinal tract which contain as active ingredient an efficientquantity of a compound defined above.

Preferred compounds are of the formula

wherein

n designates zero or an integer,

m designates 2, 3 or 4; and where

Z¹ and Z², which may be identical or different, each designates a sidechain of an amino acid.

The compounds defined above are aminoacyl derivatives when n is zero,and they are peptidyl-bisphosphonates when n is an integer as definedabove.

A wide range of amino acids can be used, and preferred ones for use inthe peptidyl chain according to the invention are:

proline

phenylalanine

alanine

lysine

arginine

aspartic acid

glutamic acid

The invention furthermore relates to a method for the production ofderivatives defined above, or their salts which comprises linking adesired bisphosphonate compound, of the Pamidronate or Alendronate type,to one or more amino acids.

The invention further relates to pharmaceutical compositions, for oraladministration, which contain an effective quantity of a novelderivative defined above. The compositions according to the presentinvention are characterized by high absorption from the gastrointestinaltract.

The dosage in humans is dependent on various factors including drugpotency, age, disease type and state, and the mode of administration.Since the present invention provides significantly enhanced oralabsorption the dosage should be determined according to the extent ofabsorption of a specific peptidyl-bisphosphonate derivative. Therefore,the dose of the prodrug is typically about 20 to 100 times lower thanthe one usually prescribed for oral administration. One should alsorecall that the MW of a prodrug is higher than the active drug. Theeffective dosage range is form about 0.001 mg to about 100 mg perpatient per day, a preferred range being of the order of about 0.02 mgto 1 mg per patient, per day in oral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the absorption of orally administered Pro-Phe-Pam invarious organs.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention is described by way of illustration onlywith reference to representative examples. It ought to be clearlyunderstood that the novel compounds can contain from one amino acid“elongation” and up to a multi-amino acid-residue peptidyl chain.

The novel compounds are effectively absorbed and after being absorbed,and due to enzymatic action, decomposed to provide the free active drug.It is preferred to use such peptidyl chains which are effective inbalancing the negative charges of the bisphosphonates. It is possiblethat some of the novel conjugates are effective as such in the humanbody.

Peptidyl prodrugs of clinically approved bisphosphonates are effectivelyabsorbed following oral administration. The present prodrug strategy wasbased on the rationale of neutralizing the negatively chargedbisphosphonate molecule by a positively charged amino acid, and or atthe same time making use of the peptide carrier system serving as atransporter for the prodrug.

Following membrane transport, the prodrug is subsequently hydrolyzed bya mucosal cell cytosolic enzyme such as prolidase, prolinase,dipeptidase, aminotripeptidase or possibly other hepatic/plasma enzymes,or is effective as such.

The rationale for the synthesis of peptidylbisphosphonates is twofold:

a) a peptidylbisphosphonate can be recognized by the nonspecific peptidetransporter, and

b) the free amino groups on the amino acid side chain are expected toneutralize partially or fully the phosphonate negative charges. Aminoacids and peptides were linked to geminal-aminoalkylidenebisphosphonates(for example Pamidronate and Alendronate) by a simple chemical procedureto afford aminoacyl—and peptidylbisphosphonates. Aminoacyl or peptidylbisphosphonates thus obtained are reconverted to the parent drug byenzymes or alternatively may be active as such in bone diseases.Synthesis of a representative example:

L-Prolyl-L-phenylalanylpamidronate (Pro-Phe-Pam,).

Experiments were also carried out with certain compounds according tothe invention, having a heterocylclic ring in the molecule.

Results similar to the ones demonstrated above and in the Figure wereobtained with these.

FIGS. 1A-1C relate to the concentration of Phe-Pamidronate andPamidronate in various organs 24 hours after Peroral Administration inrats, (Pro-(³H)Phe-(¹⁴C) Pam, (¹⁴C) Pam, 10 mg/kg. The scale indicates %of total dose.

“1” related to tibia, “2” relates to kidney, liver, intestine, “3”relates to kidney and liver.

“4”: F. Wingen and D. Schmahl, Arzneim. Forschung 37, 1037-1042 (1989).

What is claimed is:
 1. A compound represented by the structure havingthe following formulation:

on a salt thereof wherein X=H, OH n is an integer of 2 or 3 m is aninteger of 2 or 3 R is the side chain of a naturally-occurring aminoacid, and R1 is H, or R and R1 together with the NH—CH to which they arebonded represent the pyrollidine group of proline.
 2. A compoundrepresented by the structure having the following formulation:

on a salt thereof.
 3. A compound represented by the structure having thefollowing formulation:

on a salt thereof.
 4. A composition comprising the compound of claim 2,and a pharmaceutically acceptable carrier.
 5. A pharmaceuticalcomposition comprising the compound of claim 3, and a pharmaceuticallyacceptable carrier.
 6. A composition comprising the compound of claim 1,and a pharmaceutically acceptable carrier.
 7. A method of increasing theoral bioavailability of an aminoalkylidene bisphosphonate of the formula

comprising (a) coupling the aminoalkylidene bisphosphonate to adipeptide or tripeptide to form a compound according to claim 1, and (b)administering the compound according to claim 1 to a subject in needthereof, for a time and under conditions effective to achieve release ofthe aminoalkylidene bisphosphonate from said compound.
 8. A method ofinhibiting bone resorption comprising administering a compound accordingto claim 1 to a subject in need thereof, for a time and under conditionseffective to achieve release of the aminoalkylidene bisphosphonate[H₂N—(CH₂)_(n)—C(X)(PO₃H₂)₂] from said compound.